Polymerase Chain Reaction

Optimize the annealing step by increasing the temperature in 2–5 degrees C increments, and minimizing the annealing time. You can try higher annealing temperatures in the first few cycles, and lower annealing temperatures in the subsequent cycles.

Optimize the magnesium concentration for each template and primer combination.

Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR. [Lee (1995) BioTechniques 18:225].

Not enough enzyme was used.

Denaturation/extension temperature was too high and enzyme died prematurely.

Too much DMSO (>10%).

Incorrect annealing temperature: run a series of reactions using different annealing temperatures, starting 5 degrees below the calculated Tm.

Too few cycles.

Insufficient or too much Mg2+.

Poorly designed primers: double check primer sequence against template sequence, primers should have similar melting temperatures, avoid complementary sequences at the 3’ end of primers.

Carryover inhibitors (e.g., blood, serum).

Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.

Not a long enough extension time was used depending on the size of product being amplified.

Use of super-irradiated (treated with >2500 mj/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].

Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6–8% DMSO or 5% DMSO with 1.2–1.8 M betaine can be used to amplify GC-rich templates [Baskaran (1996) Genome Res 6:633].

Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin, etc.). Add BSA to the PCR (~160–600 μg/mL), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor. The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, fatty acid–free, alcohol-precipitated BSA, or Fraction V BSA all should be effective.

Incomplete denaturation (time and temperature must be long and high enough).

Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR].

10% DMSO partially inhibits Taq.

Hemin (in blood samples) inhibits Taq.

Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].

Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR [Lee (1995) BioTechniques 18:225].

Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the amount of Taq, and/or increase the volume of the PCR to dilute out them inhibitor.

- Increase the extension time from 1 min/kb to 1.5 min/kb.
- Vary total PCR cycles from 20-40; 35 cycles is typical.
- Try denaturing at 95 degrees C for 45 seconds.
- GC-rich or problematic targets work better with MgSO4 instead of MgCl2.
- For GC-rich templates, test higher annealing temperatures starting with the temperature that is equal to your primer Tm and in increments of 2 degrees C for up to six different temperatures.

- Increase the extension time from 1 min/kb to 1.5 min/kb.
- Vary total PCR cycles from 20-40; 35 cycles is typical.
- Try denaturing at 95 degrees C for 45 seconds.
- Increase the amount of template for targets >5 kb.
- Use 2.5 U of Platinum™ Taq for each 50 µL reaction.
- GC-rich or problematic targets work better with MgSO4 instead of MgCl2.
- For GC-rich templates, test higher annealing temperatures starting with the temperature that is equal to your primer Tm and in increments of 2 degrees C for up to six different temperatures.
- Vary the KB extender solution from 1.5 µL to 4.5 µL per 50 µL reaction.

Primers and Oligos

The scale that is ordered refers to the starting synthesis scale, or amount of starting material used to create your oligo. Based on purification and efficiency, you will receive less than the starting synthesis scale. However, we do have a minimum yield guarantee based on the starting synthesis scale which can be found here.

The oligo may not have been fully solubilized. After addition of TE buffer, make sure the oligo was vortexed for a full 30 seconds and/or pipette up and down more than 10 times. Primers may be present along the sides of the tubs, so when resuspending the oligo, the sides of the tubes should be “rinsed” too.

It is important to differentiate naturally occurring mutations linked to the chemical nature of the oligo manufacturing process from the perceived mutations that occur when desalted oligos are used in certain applications.

Naturally occurring mutations are inherent to the chemical synthesis of oligos and the chances of having one single insertion or deletion in a given oligo of about 30 bases is around 2%. Invitrogen will be happy to replace any oligo that falls into this category.

With regards to the perceived mutations, following DNA synthesis, the completed DNA chain is released from the solid support by incubation in basic solutions such as ammonium hydroxide. This solution contains the required full-length oligo but also contains all of the DNA chains that were aborted during synthesis (failure sequences). If a 30-mer was synthesized, the solution would also contain 29-mer failures, 28-mer failures, 27-mer failures, etc. The amount of failure sequences present is influenced by the coupling efficiency. For an oligo of this type, the percentage of full-length oligo would be between 74 and 54%, assuming a 99 or 98% coupling efficiency. This percentage is even lower when you consider oligos that are longer.

Because the oligos are synthesized from 3′ to 5′ end, the primers that are desalted and not purified for length will have missing bases at the 5′ end. Hence, oligos that are desalted are only recommended for diagnostic PCR, microarray, or sequencing. Invitrogen recommends purification of the oligos if they will be used in certain demanding applications such as mutagenesis or cloning, especially if restriction sites are added to the 5′ end.

Other sources of perceived mutations for both desalted and purified oligos are sequencing artifacts, point mutations introduced during PCR, unstable stem-loop structures in the primers, propagation of the plasmid DNA after cloning in an E. coli strain, i.e., muS, mutD, or mutT or a silent mutation selected by the bacterial strain because of codon usage in that strain.

Better purification of the oligos is recommended to provide you with full-length oligo sequence. Adding restriction sites adds on 10 or more bases to the basic 20–25-mer, making primers longer than 30 bases with a relatively low percentage of full-length sequences after desalting. Additionally, failure sequences occur at the 5′ end of the sequence as oligos are generated from 3′ to 5′ end. Therefore, restriction sites introduced at the 5′ end of primers can be compromised, resulting in missing bases.

If detritylation occurs inappropriately and/or if the synthesizer has an error and delivers the wrong base, an extra inserted base can occur in your primer. Please contact techsupport@lifetech.com for assistance.

Most of the time the color should not affect PCR or any other experimental application since typically it is caused by the iodine used in the synthesis. There are some exceptions, however. Brown oligos can also be caused by the primer being overdried, and if this is the case, the primer may not work.

If the oligo was overheated, it will appear as a “ball”-shaped pellet attached to the bottom of the tube. This should not affect the quality of the oligo, and the oligo should be readily soluble in water.

Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.

Oligos should be run on a polyacrylamide gel containing 7 M urea and loaded with a 50% formamide solution to avoid compressions and secondary structures. Oligos of the same length and different compositions can electrophorese differently. dC’s migrate fastest, followed by dA’s, dT’s, and then dG’s. Oligos containing N’s tend to run as a blurry band and generally have a problem with secondary structure.